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Environmental and Experimental Botany 77 (2012) 1– 11

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

journa l h omepa g e: www.elsev ier .com/ locate /envexpbot

hort-term responses of Picea asperata seedlings of different ages grown in twoontrasting forest ecosystems to experimental warming

henfeng Xua,b, Huajun Yina, Pei Xionga, Chuan Wana, Qing Liua,∗

Institute of Ecological Forestry, Sichuan Agricultural University, Chengdu 611830, ChinaChengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China

r t i c l e i n f o

rticle history:eceived 26 May 2011eceived in revised form 7 September 2011ccepted 21 October 2011

eywords:pen top chamberoil N turnoveroniferous foresthenologyrowthhotosynthesis

a b s t r a c t

Low temperatures are generally limiting factors in alpine ecosystems. Predicted global warming thereforecould have profound impacts on these ecosystems in the future. This study was conducted to compareeffects of experimental warming on the phenology, growth and photosynthesis of dragon spruce seedlingsof two age classes (2- and 8-year-old seedlings) grown in two contrasting subalpine forest ecosystems(dragon spruce plantation versus spruce-fir dominated natural forest) using the open top chamber (OTC)method in the Eastern Tibetan Plateau of China. The OTCs enhanced daily mean air (30 cm above the soilsurface) and soil temperatures (5 cm below the soil surface) by 1.2 ◦C and by 0.6 ◦C in two experimentalsites, respectively, throughout the growing season. Conversely, soil volumetric moisture declined by 3.8%and 2.8% in the plantation and natural forest. Experimental warming markedly extended the growingseason of two age classes in both sites. However, there were no clear differences in phenology betweenages or sites. Warming often increased the growth and photosynthesis of dragon spruce seedlings. Therewere pronounced differences in the morphological and physiological performances between ages orsites. Nevertheless, there were no significant interactions of warming, age and site on phenology, growthand photosynthesis. Irrespective of seedling ages or experimental sites, artificial warming had significantincreases in component biomass except the root. The size of warming effect on biomass depended stronglyon seedling age and experimental site. Elevated temperatures remarkably increased net N mineralizationrates and extractable inorganic N pools in both sites. Both net N mineralization and extractable inorganic

N pool were pronouncedly greater in the natural forest than in the plantation. Taken together, our resultsindicate that warming generally has positive effects on the phenology, growth and photosynthesis forboth seedling age classes in any site. Younger seedlings are more sensitive to warming as compared withthe older seedlings. Reforestation dramatically affects the responses of soil N turnover and availability towarming. We conclude that both direct and indirect warming effects synchronously modify the seedling responses.

. Introduction

Global air temperatures are predicted to increase 1.8–4.0 ◦Cver this century, with a greater warming occurring in the higheratitudinal and altitudinal ecosystems (IPCC, 2007). With the tem-erature effects on almost all biochemical processes, projectedlobal warming can directly affect the physiological performancesnd growth rates of vegetations (Llorens et al., 2004; Zhao and

iu, 2008). Moreover, global warming can also indirectly influencehe plant physiological mechanisms and growth through alteringhe growing seasons, changing soil water content and increasing

∗ Corresponding author at: Chengdu Institute of Biology, Chinese Academy of Sci-nces, No. 9 Section 4, South Renmin Road, P.O. Box 416, Chengdu 610041, China.el.: +86 28 85238944; fax: +86 28 85222753.

E-mail address: liuqing@cib.ac.cn (Q. Liu).

098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2011.10.011

© 2011 Elsevier B.V. All rights reserved.

soil N turnover and availability (Wan et al., 2005; Kudernatschet al., 2008). Low temperatures, low nutrient availabilities and shortgrowing season are generally considered to be the most impor-tant limiting factors controlling the performance of alpine plants(Gugerli and Bauert, 2001; Hyvönen et al., 2007). Such projectedwarming therefore has great potential to influence the establish-ment, survival, productivity of alpine vegetation.

In general, leaf morphology and physiology vary with treeage (Day et al., 2001; Thomas and Winner, 2002; Mediavillaand Escudero, 2003). Furthermore, many previous studies havedemonstrated that juvenile age trees may be more sensitive to envi-ronmental changes (i.e., temperature, moisture) than mature trees(Howe et al., 2004; Greenwood et al., 2008), with less research

focusing on the ecophysiological performances of younger, andsmaller life stages (Reinhardt et al., 2009). Comparative studiesof different growth stages may provide essential information forunderstanding the strategies adopted by the species at different

2 d Experimental Botany 77 (2012) 1– 11

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tages of their life cycle, as well as the selective pressures thatperate in each stage (Mediavilla and Escudero, 2003). To ournowledge, to date, little is known about warming effects on theorphological and physiological performances of the trees with

ifferent life stages.The sub-alpine and alpine forest ecosystems in the eastern

ibetan Plateau located at the transitive zone from Qinghai-Tibetlateau to Sichuan basin could be very sensitive to global climatehange with important consequences for the global C and N bal-nce (Wang et al., 2003). Last century, natural coniferous forests inouthwestern China were deforested and reforested with dragonpruce (Picea asperata Mast.). Currently, there are over 1 millionectares of dragon spruce plantation in western Sichuan, account-

ng for approximately fifty percent of the forest area in this region.he magnitude of warming on the Tibetan Plateau is projected toe large relative to many other regions and the soils on the Tibetanlateau contain large amounts of soil organic matter (Xu et al.,003; Jiang et al., 2009). Thus, the soil N turnover and growth oflpine forest in this region could be more susceptible than in othercosystems under future warmer conditions.

Ecosystem response could depend strongly on initial conditionsf ecosystems, such as stocks and initial turnover rates of soilrganic matter, the dominant form of available N in the soil, thehemical composition and turnover rates of plant residues (Shavert al., 2000). Soil responses to warming are likely to be compli-ated by land-use change (Striegl and Wichland, 1998; Zhang et al.,005). Reforestation is one of the most important land-use prac-ices. This practice often has profound impacts on the soil propertiesf forest ecosystems. Thus, reforestation could largely affect theesponses of soil ecosystems to projected global warming, and fur-her influence the vegetations growing in these soils. Therefore, its very important to investigate synchronously soil properties andeedling growth in different land-use types in a warming experi-ent, particularly in the early stages. In addition, to our knowledge,

ess emphasis has been placed on the comparison of the eco-hysiological performances of different age classes under warmingonditions. Such information could be helpful to understand theegeneration and establishment of plants under a warmer world,nd to accurately predict responses of terrestrial ecosystems touture global warming. In this study, we used open top chamberOTC) technique to simulate global warming. This experiment wasonducted to investigate the effects of experimental warming onhe growth and photosynthesis of dragon spruce seedlings of twoge classes grown in two contrasting forest soils (dragon sprucelantation versus spruce-fir dominated natural forest). These datallow us to: (1) examine how seedlings growth and soil N turnoveresponse to experimental warming in both ecosystems; (2) identifyhether there are response differences in plant growth and soil N

tatus between sites under the warmer conditions; and (3) examinehether there are differences in plant responses between ages.

. Materials and methods

.1. Experimental site

The study was conducted on two sites that were within approx-mately 300 m distance of each other (Fig. 1). One site was in aragon spruce plantation (65-year-old) and the other was in a

able 1oil organic C (SOC), total N, P and K, C/N, and pH for soils in both dragon spruce plantatio

Forest type SOC (g kg−1) Total N (g kg−1) Total P (g

Plantation 50.24(3.83)a 2.95(0.44)a 0.65(0.03Natural forest 145.02(14.87)b 9.56(1.19)b 0.67(0.07

alues within the same column with different letters are significantly different at P < 0.05

Fig. 1. Location of the study site in Miyaluo area of western Sichuan.

spruce-dominated natural forest (200-year-old). There is approx-imately 500 m from our study site to tree line. Both experimentalsites are located at the Miyaluo Experimental Forest of LixianCounty, Eastern Tibetan Plateau (31◦35′N; 102◦35′E; 3150 m a.s.l).This dragon spruce plantation was originated from the clear-cuttingland of 1940s. The climate belongs to a montane monsoon thatis humid and rainy in the summer but cold and dry in the win-ter. The mean annual temperature is 6–10 ◦C, with a maximummonthly mean air temperature of 12.6 ◦C in July and a minimum of−8 ◦C in January. Mean annual precipitation ranges from 600 mmto 1100 mm. Soils at two sites are classified as the mountain brownsoil series (Chinese taxonomy). Basic soil properties are shown inTable 1.

2.2. Open top chamber installation

In order to increase the air temperature, in late September 2008,six open top chambers (OTCs) were set up in two contrasting forestsites with similar canopy, respectively. One control plot was ran-domly established in the vicinity of each OTC. The OTCs used in thisstudy was made of acrylic glass, which transmits approximately90% of photosynthetically active radiation. The OTCs was hexago-nal and 80 cm high, with 2.45 m2 in the ground area and 1.64 m2 inopen-top area. It was expected that all of the selected plots weresimilar in microhabitat characteristics.

2.3. Microclimate monitoring

To quantify the environmental factors affected by the OTC, twoautomatic recording systems were set up in both experimentalsites, respectively. Air temperature at 30 cm high and soil temper-ature 5 cm below the soil surface were measured by alternatingamong sensors connected to a dataloger (Campbell AR5, Avalon,USA). Temperature monitoring was carried out in three OTCs andthree control plots, respectively. Data were taken at 60 min inter-vals with the automatic recording system from May to October2009.

2.4. Plant material

In our study region, 2- and 8-year old seedlings are very cru-cial two stages in the life history of dragon spruce. Therefore,

n and natural forest ecosystems.

kg−1) Total K (g kg−1) C/N pH

)a 14.16(0.39)a 17.03(1.05)a 6.19(0.47)a)a 11.31(0.51)b 15.16(0.87)b 5.85(0.65)a

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niform and healthy younger (2-year-old) and older (8-year-old)ragon spruce seedlings (Picea asperata Mast.) from a local nurs-ry were selected based on plant height, basal diameter and fresheight. Seedlings were transplanted into the OTCs and controllots. Each OTC or control plot was divided into four 40 cm × 40 cmubplots. Two diagonal subplots in each plot were used for planting-year-old seedlings; the other two were used for planting 8-year-ld seedlings. Five 8-year-old and nine 2-year-old seedlings werelanted in each subplot, respectively. The seedlings were trans-lanted in late September 2008 and the observations were initiatedrom late April 2009, providing more than seven months equilib-ium period to minimize the transplanting disturbance.

.5. Phenology observation

In order to observe phenological events, six plants in each agelass were randomly chosen in each plot, and marked with smalllastic taps before bud burst in April 2009. Bud phenology (e.g., budreak, bud set) was observed at approximately three-day interval.n this study, the beginning of bud burst was calculated at the timef 50% of bud break on the marked plants. Similarly, the beginningf bud set was calculated at the time of 50% of bud coloring on thearked branches. The growing season was defined as the interval

anging from bud break to bud dormancy.

.6. Growth measurement

After planted, each seedling was tagged and its basal diameternd height were measured and recorded. At the end of growingeason, basal diameter and height were measured again. Relativencrements in basal diameter or height were then calculated for onerowing season separately as (valueOct − valueApr)/valueApr × 100.ikewise, we measured the length of current shoot originating fromhe top bud on tagged plants. At the end of growing season, 30andomly selected seedlings (5 plants per plot) in each age classere destructively sampled to determine the stem, foliage, and root

iomass. Plant fractions were oven-dried separately for 72 h at 80 ◦Cnd the dry mass of each fraction were determined. Dried leavesere pooled and ground to determine the total nitrogen contentsing UDK152 apparatus (Velp Scientifica, Ulpiate, Milan, Italy).ewly produced leaves were digitally scanned into a personal com-uter and then analyzed with a UTHSCSA ImageTool (IT) analysisystem (University of Texas Health Science Center, San Antonio,X, USA) to determine the leaf area. The leaves were then dried to

constant mass at 70 ◦C. The specific leaf area (SLA) was calculatedfter the dry weight was determined.

.7. Gas exchange

A portable photosynthesis system (Model LI-6400; LI-COR,nc., Lindoln, Nebraska) in open-circuit mode was used to deter-

ine instantaneous gas exchange on fully expanded current-yeareedles under controlled optimal conditions. The photosyn-hetic photon flux density (PPFD) was maintained at 1000 �molphoton) m−2 s−1 using the LI-6400 artificial light source, and tem-erature was maintained at 20 ◦C with a relative humidity of 60%

nside the leaf chamber during measurement. The CO2 concen-ration within the leaf measurement chamber was maintained at80 �mol m−2 s−1. Net photosynthetic rate (A), transpiration rateE), stomatal conductance (gs) were measured. Leaves used forhotosynthetic measurements were scanned and analyzed with

UTHSCSA ImageTool (IT) analysis system (University of Texasealth Science Center, San Antonio, TX, USA) to determine the leafrea. All of the measurements were completed over 7 days, from9:00 to 11:30 each day.

rimental Botany 77 (2012) 1– 11 3

Light response curves of the fully expanded current-yearneedles were measured at 0, 20, 50, 80, 100, 150, 200, 400,600, 800, 1000, 1200, and 1500 �mol (photon) m−2 s−1 of photo-synthetic active radiation under the uniform conditions (20 ◦C,380 �mol (CO2) m−2 s−1, and 60% RH). The linear regressions of PARand A over the range of 0–200 �mol (photon) m−2 s−1 of PAR wereapplied to determine apparent quantum yield (AQY). Maximum netassimilation rate (Amax), light compensation point (LCP) and darkrespiration rate (Rd) were estimated according to Ye (2007).

2.8. Soil sampling and analysis

Soil samples were collected from the topsoil (0–15 cm) late inthe growing season of 2009. One core (3 cm in diameter, 0–15 cmdeep) was randomly taken in each subplot. The four soil cores fromeach plot were mixed to get one composite sample and deliveredimmediately to the laboratory. Each composite sample was passedthrough a sieve (4 mm diameter), and any visible living plant mate-rial was manually removed from the sieved soil. The sieved soil waskept in the refrigerator at 4 ◦C for soil analysis. Extractable inorganicN (ammonium and nitrate) was extracted with a 2 M KCl extractingwater solution. Ammonium and nitrate in extract was measured bycolorimetry.

Soil N mineralization rates were measured from in situ incu-bations using the buried bag technique (Adams et al., 1989). Theincubations were performed using perforated PVC tubes (15 cm inheight and 5 cm in diameter). Parafilm covered the top of each tubeto avoid leaching of nitrate. The technique prevents plant uptakeof mineralized nutrients but allows uptake by the microorganisms.The seasonal net N mineralization was expressed as the differencein inorganic N (nitrate and ammonium) in the soil before and afterincubation in the bags.

2.9. Statistical analysis

Three-way analysis of variance (ANOVA) was used to exam-ine the effects of warming, site, age and their interactions forphenological events (bud break, bud dormancy and growing sea-son), growth parameters (specific leaf area, relative increment inbasal diameter and height), and photosynthetic parameters (A, E,gs, Amax, LCP, Rd, AQY and leaf N content). For specific param-eter noted in this study, Student t-tests were used to comparethe effect of the experimental warming. In order to assess ageand site effects on plant biomass, the effect size (ES) is definedas: ES = ln(MeanOTC/Meancontrol), where MeanOTC is the mean forone OTC (five plant), Meancontrol is the mean for one control plot(five plant). Values above zero indicate a positive effect (warmingcaused an increase in the biomass). Two-way analysis of vari-ance (ANOVA) was used to examine the effects of age, site andtheir interactions for effect size. Two-way analysis of variance(ANOVA) was also conducted to examine the effects of site, warm-ing and their interaction for soil N mineralization and inorganicN pool. Before analysis, all data were tested for the assumptionsof ANOVA. If data were heterogeneous, they were ln-transformedbefore analysis. The statistical tests were considered significant atthe P < 0.05 level. All statistical tests were performed using the SPSS13.0.

3. Results

3.1. Microclimate

The OTCs caused expected warming effects in both experimen-tal sites. In contrast to the control plot, air temperature and soiltemperature in the OTCs were on average increased by 1.2 and0.6 ◦C in both experimental sites throughout the growing season

4 Z. Xu et al. / Environmental and Experimental Botany 77 (2012) 1– 11

Table 2Monthly mean air temperature (◦C), soil temperature (◦C) and soil moisture (%) in the open top chambers and control plots of two experimental sites.

Forest type Month Air temperature Soil temperature Soil moisture

OTC Control plot OTC Control plot OTC Control plot

Natural forest May 10.8 9.7 9.6 9.2 25.5 28.7June 11.8 10.6 10.8 10.3 28.0 29.7July 14.2 12.9 13.0 12.2 29.9 33.5August 14.3 13.1 13.1 12.5 27.3 30.8September 13.3 12.2 12.8 12.2 29.6 31.4October 6.3 5.1 7.9 7.5 36.2 39.1

Plantation May 10.5 9.4 9.6 9.1 25.1 31.9June 11.9 10.6 10.9 10.3 25.8 30.0July 14.8 13.3 13.4 12.6 26.9 30.4

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Table 2). Conversely, soil volumetric moisture at 10 cm deptheclined 3.8% in the plantation and 2.8% in the natural forest,espectively, throughout the growing season (Table 2). Moreover,here was no obvious seasonality in soil moisture in both forestcosystems (Table 2).

.2. Bud phenology

Irrespective of experimental sites or seedling ages, artificialarming had remarkable effects on the phenological process of

he dragon spruce seedlings (Tables 3 and 4). There was significantifference in bud break between warming regimes regardless ofites or ages (Table 3). Either 2- or 8-year-old seedlings grown inhe OTCs showed significantly later bud dormancy in both experi-

ental sites. Warming significantly extended the growing seasonength of the dragon spruce seedlings (Table 3). Statistical analyseshow that seedling age and experimental site had no clear effectsn bud phenology (Table 4). Likewise, no any interactive effect onud phenology was detected (Table 4).

.3. Growth parameters

Regardless of seedling ages or experimental sites, elevated tem-

eratures significantly increased the relative growth of plant heightnd basal diameter, current shoot length and specific leaf areaFig. 2; Table 4). Additionally, there was significant difference inelative increment of plant height and basal diameter, and specific

able 3he differences (days) of phenological events between the open top chambers and contro

Forest type Seedling age Bud break

Plantation 2 +6.8**

8 +8.2**

Natural forest 2 +7.3**

8 +7.7**

+” advanced; “−”delayed; “↑”extended.* P < 0.05.

** P < 0.01.

able 4esults of three-way ANOVA showing the P values for responses of bud break, bud dorman

eaf area to warming (W), site (S), and age (A).

Factor Bud break Bud dormancy Growing season

Age 0.195 0.168 0.187

Site 0.368 0.327 0.348

Warming <0.001* <0.001* <0.001*

A × S 0.323 0.286 0.308

A × W 0.349 0.278 0.317

S × W 0.290 0.236 0.252

A × S × W 0.447 0.335 0.385

* P < 0.05.

13.6 12.9 28.1 31.213.1 12.7 30.4 33.3

7.5 6.8 35.5 37.8

leaf area between 2- and 8-year-old seedlings (Table 4). Similarly,experimental site also pronouncedly affected the relative growth inplant height and basal diameter, but did not influence the specificleaf area (Table 4). Statistical analyses indicate that there was noany interactive effect of warming, site and age on growth parame-ters (Table 4).

3.4. Biomass accumulation

Irrespective of seedling ages or experimental sites, artificialwarming tended to cause significant increases in componentbiomass (Fig. 3A–D). Experimental warming significantly increasedleaf and total biomass for both seedling age classes at both sites(Fig. 3A–D). Similarly, elevated temperatures induced a significantincrease in stem biomass except the younger seedlings grown in theplantation site (Fig. 3A–D). Conversely, artificial warming did notobviously influence root biomass for 2- and 8-year old seedlings inany site (Fig. 3A–D). Statistical analyses indicate warming effects onbiomass, to some extent, depended on experimental site or seedlingage (Fig. 4). Additionally, site and age alone significantly influencedthe performances of stem and total biomass under the warmingconditions (Fig. 4).

3.5. Photosynthetic parameters

Irrespective of seedling ages, artificial warming tended tostimulate the gas exchange of dragon spruce seedlings in both

l plots in both dragon spruce and natural forest.

Bud dormancy Growing season

−6.1** ↑12.9**

−5.6* ↑13.8**

−5.8** ↑13.1**

−4.8* ↑12.5**

cy, growing season, relative growth rate (RGA) in diameter and height and specific

RGR in diameter RGR in height Specific Leaf area

<0.001* <0.001* 0.007*

0.008* 0.012* 0.4830.002* <0.001* <0.001*

0.365 0.323 0.8390.647 0.526 0.7400.479 0.428 0.8190.959 0.756 0.872

Z. Xu et al. / Environmental and Experimental Botany 77 (2012) 1– 11 5

F , curreg values

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ig. 2. Effects of warming on relative increments in plant height and basal diameterrown in the plantation and natural forest soils. Student t-test, *P < 0.05, **P < 0.01;

xperimental sites (Figs. 5A–D and 6A–D). Elevated tempera-ures significantly increased the instantaneous net photosyntheticate (A), transpiration rate (E), stomatal conductance (gs), max-mum net photosynthetic rate (Amax), apparent quantum yieldAQY), dark respiration rate (Rd), photosynthetic light compensa-ion point (LCP) and leaf N concentration for both age seedlingsn each experimental site (Figs. 5A–D and 6A–D). Furthermore,

eedling age had significant effects on gs, E, LCP, Rd, AQY andarginally significant effects on A, Amax (Table 5). Similarly,

he effects of experimental site were significant on E, AQY,nd leaf N concentration, but not significant on A, gs, and LCP

able 5esults of three-way ANOVA showing the P values for responses of leaf N concentration, neuantum yield (AQY), maximum net assimilation rate (Amax), light compensation point (L

Factor Leaf N A gs E

Age 0.121 0.088** 0.003* 0.001Site 0.002* 0.117 0.132 0.035Warming 0.013* 0.008* 0.001* <0.001A × S 0.973 0.880 0.582 0.707A × W 0.363 0.398 0.551 0.549S × W 0.837 0.318 0.742 0.626A × S × W 0.995 0.599 0.945 0.941

* P < 0.05.** P < 0.1.

nt shoot length, and specific leaf area of 2- and 8-year-old dragon spruce seedlings are means ± S.D.

(Table 5). Whereas, no any interactive effect of warming, siteand age on gas exchange and leaf N concentration was detected(Table 5).

3.6. Soil N mineralization and availability

In the incubation experiments with the absence of plants, warm-

ing induced a remarkably higher net N mineralization rate in theplantation. Likewise, similar tendency was also observed in the nat-ural forest (Fig. 7A). In addition, there was significant difference insoil N mineralization between two contrasting sites. However, the

t photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), apparentCP) and dark respiration rate (Rd) to warming (W), site (S), and age (A).

Amax LCP Rd AQY

* 0.073** 0.002* 0.019* <0.001*

* 0.091** 0.148 0.063** 0.048*

* 0.011* 0.006* <0.001* <0.001*

0.557 0.616 0.822 0.221 0.878 0.96 0.686 0.933 0.637 0.799 0.643 0.650 0.930 0.944 0.988 0.978

6 Z. Xu et al. / Environmental and Experimental Botany 77 (2012) 1– 11

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Fig. 3. Effects of warming on biomass (dry mass) accumulation of 2- and 8-year-old dragon spruce seedlings grown in the plantation and natural forest soils. Student t-test,*P < 0.05, **P < 0.01; values are means ± S.D.

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ig. 4. Effect size in the biomass of 2- and 8-year-old dragon spruce seedlings growA: seedling age effect, FS×A: interaction effect of experimental site × seedling age.

nteractive effect of warming × site was not significant on soil Nineralization (Fig. 7A).Experimental warming significantly increased the extractable

norganic N pool in both sites (Fig. 6B). Extractable inorganic Nool was markedly higher in the natural forest than in the plan-ation (Fig. 6B). Furthermore, the interaction of site × warming was

arginally significant on the extractable inorganic N pool (Fig. 6B).

e plantation and natural forest soils. Two-way ANOVA; FS: experimental site effect,

4. Discussion

4.1. Direct warming effects

4.1.1. Direct warming effect through stimulating growth rateTemperature almost affects all biochemical processes in terres-

trial ecosystems. Therefore, global warming is likely to influence

Z. Xu et al. / Environmental and Experimental Botany 77 (2012) 1– 11 7

Fig. 5. Effects of warming on net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs) and leaf N concentration of 2- and 8-year-old dragon spruces , **P <

g dlings

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eedlings grown in the plantation and natural forest soils. Student t-test, *P < 0.05ray bar: 2-year-old seedlings grown in the control plots; black bar: 8-year-old see

lant growth through direct effects on metabolic rates, such as pho-osynthesis, respiration, and nutrient and water uptake (Llorenst al., 2004). Additionally, temperature is a key factor that regu-ates ontogenetic development rate (Morison and Lawlor, 1999).revious studies have reported that experimental warming accel-rated organ initiation and expansion rates (Wada et al., 2002;lorens et al., 2004; Xu et al., 2009). In the present case, artifi-ial warming stimulated the growth of 2- and 8-year-old seedlingsegardless of experimental sites, judged from the increased planteight, basal diameter, current shoot length and biomass in theTCs. Irrespective of experimental sites, elevated temperaturesroduced significant increases in the SLA for two age classes. Simi-

ar response was also found in other coniferous tree seedlings (Zhat al., 2002; Zhao and Liu, 2008). However, this result was differ-nt from the performances of rose hyvrida and dwarf shrubs in

middle-latitude alpine region (Suzuki and Kudo, 2000; Pandeyt al., 2007). Additionally, warming pronouncedly increased theurrent shoot length regardless of seedling ages or experimentalites. Our results were consistent with those from other warmingxperiments (Wada et al., 2002; Llorens et al., 2004; Xu et al., 2009).he increased current shoot length and SLA in the warmed plotsuggest that warming treatment leaded to an increase in cell num-er or size, or both. Biomass accumulation is an important plantesponse to environmental changes. Irrespective of seedling ages orxperimental sites, warming tended to cause significant increasesn component biomass except for root biomass. This response was

ossibly because the magnitude of soil warming caused by theTCs was relatively small in both sites. Moreover, transplanting

tress may, to some extent, mask the warming effects on rootrowth.

0.01; values are means ± S.D.; blank bar: 2-year-old seedlings grown in the OTCs; grown in the OTCs; stripe bar: 8-year-old seedlings grown in the control plots.

4.1.2. Direct warming effect through enhancing photosyntheticcapacity

Low temperature stress produces a decrease in leaf gasexchange. In contrast, raising temperatures may increase theleaf size, carbon assimilation and transpiration rate in regionswhere low temperatures are limiting the physiological activities ofplants. Additionally, global warming may induce ecophysiologicalchanges in plants which could affect their long-term performances(Michelsen et al., 1996). Both photosynthesis and respiration aretemperature dependent and among the most sensitive processesin response to global warming. Photosynthesis and respirationare interdependent, with respiration relying on photosynthesis forsubstrate, whereas photosynthesis depends on respiration for arange of compounds, such as carbon skeletons for protein synthesisand ATP for sucrose synthesis and repair of photosynthetic pro-teins (Atkin et al., 2000). The temperature-mediated differences inleaf respiration are tightly linked to concomitant differences in leafphotosynthesis (Atkin et al., 2005). A change in temperature mightresult in an immediate alternation in the rate of each process. In thisstudy, elevated temperatures significantly enhanced A, Amax andRd. This was possibly because that warming provided more optimaltemperature conditions for gas exchange (Wang et al., 1995). More-over, in the present case, artificial warming significantly increasedthe leaf N concentrations for two age classes regardless of experi-mental sites. The increased leaf N concentration in the OTCs may, tosome extent, contribute the increased leaf photosynthesis. Because

leaf N concentrations are associated closely with Rubisco content,since Rubisco constitutes about 50% of leaf protein in which 70–80%of leaf N is invested (Field and Mooney, 1986). AQY is the efficiencyof light utilization in photosynthesis. In this study, experimental

8 Z. Xu et al. / Environmental and Experimental Botany 77 (2012) 1– 11

Fig. 6. Effects of experimental warming on apparent quantum yield (AQY), maximum net assimilation rate (Amax), light compensation point (LCP) and dark respiration rate( tural f2 e conts

wowe

4

4

s

Fb

Rd) of 2- and 8-year-old dragon spruce seedlings grown in the plantation and na-year-old seedlings grown in the OTCs; gray bar: 2-year-old seedlings grown in theedlings grown in the control plots.

arming also significantly increased the AQY of 2- and 8-year-ld dragon spruce seedlings. Such positive effects of experimentalarming on AQY have also been reported by other studies (Saxe

t al., 2001; Zhao and Liu, 2008).

.2. Indirect warming effects

.2.1. Indirect warming effect through altering plant phenologyIn combination with warming effects (e.g., extended growing

eason, soil nutrient availability), changes in these factors may

0

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ig. 7. Effects of experimental warming on net soil N mineralization and extractable inorgars with different letters are significantly different form each other. FW: warming effect, F

orest soils. Student t-test, *P < 0.05, **P < 0.01; values are means ± S.D.; blank bar:rol plots; black bar: 8-year-old seedlings grown in the OTCs; stripe bar: 8-year-old

also influence the growth, photosynthetic performance of plants(Wan et al., 2005; Kudernatsch et al., 2008). Growing season can beextended by early bud break, late dormancy, or combination. In thisstudy, as a result of warming effects, bud break was significantlyadvanced and bud dormancy was pronouncedly delayed regardlessof experimental sites or seedling ages. Therefore, growing season

was substantially extended by experimental warming. On theother hand, the number of days with daily mean air temperatureabove 10 ◦C was greater in the OTCs than in the control plots, alsoimplying a longer growing season in the OTCs. The phenological

0

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anic N in two contrasting forest soils. Two-way ANOVA, Values are means ± S.D. TheS: experimental site effect, FS×W: interaction effect of experimental site × warming.

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esponses noted in this study were in good agreement with manyther similar studies (Arft et al., 1999; Wada et al., 2002; Norbyt al., 2003; Xu et al., 2009). The phenological differences causedy warming could benefit gross photosynthesis and plant growthNorby et al., 2003; Wan et al., 2005). Warming-induced pheno-ogical changes would lead to a longer favorable period for planthotosynthesis and growth, resulting in increased leaf area indexnd green biomass during the growing season. In this study, thextended growing season could, at least partially, contribute thencreased growth of dragon spruce seedlings. On the other hand,

arming-induced changes in spring phenology may increase theisk of frost damage (Bokhorst et al., 2010; Taulavuori et al., 2004).owever, we did not found any obvious frost injury in this study.

.2.2. Indirect warming effect through increasing soil Nvailability

Warming-induced changes in the release of nutrients may affectlant response indirectly (Verburg and Breemen, 2000). Low tem-erature and N availability are generally considered to be the most

mportant limiting factors controlling the performance of alpinelants (Gugerli and Bauert, 2001). Temperature is key factor thategulates the N turnover and transformation of soil ecosystemsHart and Perry, 1999). Thus, projected global warming is likelyo stimulate soil N mineralization at large. Some studies haveemonstrated that soil N availability increased with soil warming.evertheless, these studies were mainly conducted in high-latitudecosystems (Rustad et al., 2001; Biasi et al., 2008; Sardans et al.,008). In this study, experimental warming caused significant

ncreases in soil N availabilities in both forest ecosystems. Addi-ionally, irrespective of experimental sites, net soil N mineralizationate was much higher in the OTCs versus in the control plots. There-ore, the increased N availability could be the result of the higher N

ineralization induced by warming. Our result was consistent withhose from other warming experiments conducted in arctic andlpine ecosystems (Rustad et al., 2001; Biasi et al., 2008). In addi-ion, the effect size of warming on N availability was relatively greatn the natural forest in comparison with the plantation. This maye explained by the differences in soil N pools and soil physical andhemical properties (relatively large bulk density, small porositynd slow decay of SOM in the plantation) between two contrastingcosystems (Liu et al., 2002). Warming-Increased N mineralizations likely to favor net primary production (NPP) especially in areas

here N is limiting NPP (Verburg and Breemen, 2000). Therefore,he increased soil N availability observed in this study could, toome extent, benefit the N uptake and growth of the seedlings.

.3. Differences in growth and photosynthesis between agelasses

Many aspects of leaf morphology and physiology may vary withree age (Thomas and Winner, 2002; Mediavilla and Escudero,003). Furthermore, most previous studies have demonstrated that

uvenile age classes are likely to be more sensitive to environmentaltress (e.g., temperature and moisture) than mature trees (Howet al., 2004; Greenwood et al., 2008), with less research focusingn the ecophysiological performances of younger, and smaller lifetages. In this case, experimental warming stimulated the growthn both age classes. Whereas, compared with the older seedlings,

arming-induced increases in growth and gas exchange were rel-tively great for 2-year-old seedlings, indicating superior warmingensitivity of younger seedlings. On the other hand, irrespective ofarming treatments, 2-year-old seedlings often exhibited stronger

erformances in growth rate and photosynthesis parameters (e.g.,GA in height and diameter, SLA, A, AQY) compared to the 8-year-ld seedlings in both experimental sites. Comparing the growthnd ecophysiological performances among different seedling ages

rimental Botany 77 (2012) 1– 11 9

may help to identify potential life-stage bottlenecks and enableearly predictions about ultimate regeneration. Compared with the8-year-old seedlings, 2-year-old seedlings allocated more assim-ilation to leaf growth. This was probably because very youngseedlings generally lack fully expanded lateral branches and maydevote more assimilation to new needle growth (Kohyama, 1983).Comparative studies of different growth stages may provide essen-tial information for understanding the strategies adopted by thespecies at different stages of their life cycle, as well as the selec-tive pressures that operate in each stage (Mediavilla and Escudero,2003). In this case, there were clear differences in photosyntheticperformances (e.g., LCP, AQY and gs) between two age classes. Thismay be due to the differences in sunlight interception among theage classes or may be indicative of developmental or physiologicaldifferences between age classes. Additionally, faster growth dur-ing the early establishment may make germinant seedlings morecompetitive, and help them to enhance their survivals. Seedlingestablishment and juvenile growth are critical periods in the lifecycle of tree species (Reinhardt et al., 2009). In our study site, inthe natural conditions, younger seedlings often face larger stress(e.g., low light, low temperature and animal feed) as comparedto the older seedlings. Because very young seedlings were muchfew in our studied forests (Yin et al., 2007). Thus, this stage maybe a key period for dragon spruce establishment and regeneration.However, the seedlings with higher SLA in the warmed plots wouldbe more susceptible to disease or pathogens attack. In our study,elevated temperatures resulted in stronger responses in youngerseedlings as compared with the older seedlings. Therefore, pro-jected global warming may, to some degree, favor the regenerationof dragon spruce forest.

4.4. Response differences in plant and soil between sites

Ecosystem response could depend strongly on initial condi-tions of ecosystems, such as stocks and initial turnover rates ofsoil organic matter, the relative size of the plant and soil C pools,the dominant form of available N in the soil (Shaver et al., 2000).Soil responses to warming are likely to be complicated by land-usechange (Striegl and Wichland, 1998; Zhang et al., 2005). Nutrientsavailability, particularly N availability, is the primary limiting fac-tor for plant productivity in most high-altitude and high-latitudeecosystems (Shaver and Chapin, 1980). In this case, N availabil-ity was much higher in the natural forest than in the plantation.Moreover, warming-induced increases in inorganic N pools wererelatively great in the natural forest as compared to the planta-tion. In the present case, irrespective of warming treatments, thegrowth and physiological parameters were significantly greater inthe natural forest than in the plantation. Many previous studieshave demonstrated that increasing soil N availability can enhancethe growth and photosynthetic mechanisms of plants (Mo et al.,2008). Therefore, the differences in seedling growth between twocontrasting soils may be partly explained by the differences in thesoil N availability.

5. Conclusion

First, present study demonstrated that experimental warminggenerally increased the growth and gas exchange of dragon spruceseedlings in both experimental sites. Also, seedling age and experi-mental site alone often had pronounced effects on ecophysiologicalperformances of dragon spruce seedlings regardless of warming

regimes. Nevertheless, warming effects on the growth and pho-tosynthetic parameters noted in this study did not depend onexperimental site or seedling age. On the other hand, irrespective ofexperimental sites and seedling ages, artificial warming generally

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roduced significant impacts on the component biomass of dragonpruce seedlings. Moreover, responses of component biomass tortificial warming depended partly on seedling ages and experi-ental sites.Second, elevated temperatures greatly influenced the phenolog-

cal events of both seedling age classes grown in both forest soilsut no clear difference in phenology was detected between exper-

mental sites or seedling ages. Obviously, phenological activities ofnderstory dragon spruce were not associated to life stages or soilroperties. Phenological events of understory dragon spruce maye mainly controlled by temperature.

Third, this study has also demonstrated that warming sig-ificantly increased net soil N mineralization in both forestcosystems. Additionally, higher exactable inorganic N pool waslso observed in the warmed plots, indicating that experimentalarming increased soil N turnover rate and N availability in two for-

st ecosystems. The responses of N transformation to experimentalarming depended partly on forest types. Moreover, significantifferences in N pools between two contrasting soils may, to someegree, explain the differences in growth and physiological perfor-ances of dragon spruce seedlings between sites.Finally, one growing season study could not thoroughly reflect

he responses of these seedlings to the artificial warming. However,hort-term treatment can, at least, elucidate any response patternsetween sites and ages at early stage of experimental warming.lthough a seedling study may not well-predict the performancef mature tree, physiological and morphological responses of differ-nt age classes to warming may indicate the observed results fromeedlings may, to some extent, overestimate the positive effectsf projected global warming on forest productivity. Evidence ofn inter-relation between growth and experimental site suggestshat warming impacts on tree growth could be related closely tooil properties, especially soil N status under future warming con-itions. Apparently, responses of forest ecosystems to projectedlobal warming could be greatly complicated by land-use changes.

cknowledgements

We thank the staff in the Forestry Bureau of Western Sichuanor their kind help in field investigations. This study was sup-orted jointly by the National Natural Science Foundation of China30800165, 30800161, 31000213 and 31170423), Western Lightoundation of 491 the CAS (2008) and the Talent Plan of CIB, thehinese Academy of Sciences 492 (O8B2031), New Century Excel-

ent Talents in University (NCET-07-0592), Knowledge Innovationngineering of the Chinese Academy of Sciences (Y0B2021100) andKSCX2-YW-Z1023) and National Key Technology R&D Program ofhina (No. 2011BAC09b05).

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